A very important part of our laboratory effort is to identify unique cell surface markers of KSC in order to distinguish these stem cells from other keratinocytes. Currently, unique cell surface markers have not been identified for either keratinocyte stem cells or other epithelial tissues. The ability to identify and manipulate keratinocyte stem cells is important for several reasons. In addition to allowing us to gene target these stem cells for long-term gene expression in a renewing tissue, it is possible that some of the unique markers identified for KSC may be shared by cancer stem cells and could be helpful in characterizing these cells. Our current plan is to utilize the approaches that we have developed to identify unique KSC markers can also be applied to identify cell surface markers that are only present on the cancer stem cells in skin cancers such as squamous cell carcinoma (SCC) or basal cell carcinoma (BCC). Not only would these unique markers allow us to isolate and characterize SCC/BCC cancer stem cells, but these markers may also allow therapeutic targeting of these cells. In addition, the competitive in vivo repopulation assays that we have developed to assess the KSC stem cell characteristics of self-renewal and long-term skin repopulating ability can also be utilized to assess the ability of putative cancer stem cell populations to cause SCC or BCC. Such assays would assess the ability of putative cancer stem cells to recapitulate the SCC and BCC cancers and will be critically important to prove the existence of cancer stem cells in these cancers. Ultimately, the identity and characterization of cancer stem cells may be required before curative therapies can be developed for epithelial cancers. Keratinocyte stem cells are best identified by their ability to retain a BrdU label, and we have successfully FACS-sorted pure populations of these label-retaining cells (LRC). Unfortunately, the detection of BrdU requires that cells be fixed and permeablized, making them unsuitable for biological studies requiring living cells. In order to obtain living cells for biological studies, we have tried to identify unique cell surface markers on keratinocyte LRC, using both microarray analysis of global gene expression and proteomic mass spectrometry analysis of membrane proteins. For microarray analysis, we have developed "navigated" laser capture microdissection to isolate enriched populations of LRC in the human hair follicle and have identified panels of genes, including genes that encode membrane proteins, that are differentially expressed in the LRC population as compared to non-LRC keratinocytes. In collaboration with the Biomedical Proteomics Program at FCRF, we have developed high-throughput mass spectrometry methods able to identify membrane proteins on LRC, and we are now using semi-quantitative mass spectrometry to compare the relative levels of the plasma membrane proteins prepared from LRC keratinocytes to control keratinocyte populations (transit amplifying basal keratinocytes). We have also assessed stem cell behavior in other putative KSC populations including a population of very primitive hematopoietic stem cells (side population or SP cells). These cells can be identified by unique fluorescent emission characteristics due to their ability to exclude HO33342 nuclear dye. We have recently described a SP population of keratinocytes and are currently trying to determine if SP keratinocytes possess long-term repopulating ability, using our in vivo competitive repopulation stem cell assay.